The efficiency of protein expression from genetically engineered cells is an important commercial concern. Some commercially important proteins are best made from eukaryotic cells, such as mammalian cells, plant cells, or yeast cells, in order to ensure correct folding and glycosylation. However, the cost of maintaining large cultures of eukaryotic cells means that proteins produced in this way are expensive to make. Therefore, there is a need in the art to maximize the expression levels of proteins from eukaryotic cells.
A related issue is that therapeutic proteins produced from eukaryotic cells must be expressed in the correct conformational state. Normally, mechanisms of transcription and translation ensure that a genetically engineered cell will produce a protein whose sequence is determined by the nucleic acid encoding the protein. However, after transcription and translation, the protein may fail to fold properly and may be degraded. Alternatively, a protein may be produced in an aggregated state, such that activity is reduced. Even if an aggregated protein is active, it may be pharmacologically unacceptable due to increased immunogenicity compared to a non-aggregated protein. Thus, a pharmacologically acceptable protein preparation should generally be substantially free of aggregated proteins.
The amount of a protein that is expressed from a genetically engineered eukaryotic cell is a function of the rate of transcription of the encoding gene, the efficiency of mRNA splicing and of export from the nucleus, and the efficiency of translation. The role that these events play in protein expression is sufficiently well understood that one skilled in the art of genetic engineering and protein expression can generally incorporate appropriate nucleic acid sequences into the design of an expression construct with efficient transcription, splicing, mRNA export, and translation.
However, the amount of a correctly folded, non-aggregated protein that is produced from a eukaryotic cell is also a function of the amino acid sequence of the protein, as well as the nucleic acid sequences that determine transcription, splicing, mRNA export, translation, and post-translational modification. For example, it is thought that a significant fraction of proteins synthesized in a cell is degraded. The features in a protein that determine whether or not it should be degraded are currently subject to intensive study, but presently it is not possible to predict the efficiency of protein folding, degradation, or aggregation by simply examining the sequence of a protein. Some naturally occurring proteins fold efficiently, are resistant to proteolysis, and do not aggregate. In contrast, other proteins fold inefficiently, are rapidly degraded, and aggregate.
Antibodies and artificial proteins containing a portion of an antibody, termed antibody fusion proteins or Ig fusion proteins herein, are useful for a variety of purposes relating to the targeting capability of antibody variable domains as well as the ability of the constant regions to bind to various other proteins. Antibody and antibody fusion protein preparations are particularly useful when they are correctly folded and non-aggregated. Therefore there is a need in the art for methods and compositions for the production of antibody and antibody fusion protein preparations with reduced aggregation.
Additionally, antibodies and antibody fusion proteins are useful since their ability to bind to various other proteins enables them, for example, to elicit specific effector functions. In some instances specific effector functions are desirable but often the loss of effector functions is preferable. The antibody component of a fusion protein may be altered to reduce or eliminate effector functions by utilizing a modified antibody. Antibody and antibody fusion protein preparations are also useful when they are modified to alter functionality. Therefore there is a need in the art for methods and compositions for the production of modified antibodies and antibody fusion proteins with altered effector functions.
Protein drugs can be degraded by proteases, such that their delivery and pharmacokinetic properties are suboptimal. There is a need in the art for improvement of protein drugs that have the useful properties of certain proteins, but that have greater protease resistance.